Glycolysis is required for mammalian sperm function and fertilization. Several sperm glycolytic enzymes have distinctive properties, and three isozymes in this pathway are products of genes that are expressed only in spermatogenic cells. Two of these germ cell-specific isozymes, glyceraldehyde 3-phosphate dehydrogenase-S (GAPDS) and phophoglycerate kinase-2 (PGK2), are located at a key transition point in the pathway between the two phases that consume and produce ATP. Selective inhibition of either of these isozymes would eliminate ATP production by glycolysis without affecting energy production in other tissues, indicating that GAPDS and PGK2 may be excellent contraceptive targets. Our recent studies indicate that Gapds -/- male mice are infertile, producing sperm with no progressive motility and ATP levels that are 10% of normal levels. This proposal will determine if PGK2 is similarly required for sperm motility and male fertility, and will examine the enzymatic and structural characteristics of this isozyme with the goal of identifying selective inhibitors. The specific aims are to: 1) Determine if PGK2 is required for sperm motility and male fertility. The Pgk2 gene will be disrupted in mice by homologous recombination. Fertility, spermatogenesis and sperm function will be assessed in the Pgk2 -/- animals, including assays of sperm motility, morphology and ATP levels. 2) Identify unique structural features of PGK2 and design selective inhibitors of this isozyme. Molecular modeling studies will be used to determine features that distinguish PGK2 from PGK1, the isozyme present in somatic tissues. Potential active site inhibitors will be identified by virtual screening, and the most promising candidates will be tested against recombinant PGK2 and PGK1 to identify selective inhibitors of PGK2. 3) Determine the mechanisms for PGK2 localization in sperm. Like other glycolytic enzymes, PGK2 is bound to structural components of sperm. To provide a better understanding of protein interactions that are essential for high levels of glycolytic ATP production to support sperm motility, we will determine if PGK2 is localized in the principal piece of the sperm flagellum, identify proteins that interact with PGK2, and determine if PGK2 has a specific targeting sequence that is responsible for sperm localization. This proposal will identify novel features of PGK2 and determine if this sperm isozyme is a target for developing highly specific male contraceptives.